Trypanosomatids are protozoan hemoflagellates responsible for a variety of human diseases, including Chagas'disease, leishmaniasis, and African sleeping sickness, which account for nearly one million deaths per year. The unique genetic phenomena occurring in parasites'giant mitochondria have been extensively studied, producing major advances in the understanding of kinetoplast DNA (kDNA) replication and uridine insertion/deletion mRNA editing. However, the molecular mechanisms governing mRNA stability and turnover in mitochondria remain mostly unknown. Our goal is to set the stage for molecular analysis of the regulatory mechanisms controlling mitochondrial mRNA polyadenylation, and better understanding of life cycle- correlated changes in kDNA expression. The specific hypothesis underlying the proposed research is that polyadenylation in trypanosomal mitochondria is essential for the stability of translationally-competent mRNAs and, therefore, mitochondrial function. We suggest that polyadenylation is accomplished by a Kinetoplast Poly(A) Polymerase (kPAP1) acting as catalytic subunit of multi-protein complex. KPAP1 has been identified in the course of preliminary experiments for this proposal via homology with RNA uridylyl transferases (TUTases). Our findings expand known roles of TUTase-like structural scaffolds to ATP-specific enzymes involved in mitochondrial RNA processing. We propose to: 1. Characterize the roles of kPAP1 in polyadenylation of non-edited, pre-edited, and edited mRNAs. RNA interference will be used to down-regulate expression of both kPAPs in procyclic and bloodstream forms, and their significance for mitochondrial function will be addressed by comparative analysis of cell viability, membrane potential, and RNA transcripts. 2. Elucidate the protein composition of the polyadenylation machinery in trypanosomal mitochondria. The kPAP1-containing complexes will be isolated by tandem affinity chromatography, tested for enzymatic activity, and analyzed by mass spectrometry. Narrative: Trypanosomatids are the causative agents of major parasitic diseases in developing countries around the world. Considering the occurrence of visceral leishmaniasis among U.S. troops stationed in the Persian Gulf and Afghanistan, a demand for effective anti- trypanosomal therapies also may be anticipated in the United States. Available treatments are often toxic and ineffective, creating demand for the discovery of new drugs. Targeting essential parasite-specific enzymes, such as the mitochondrial poly(A) polymerase described for the first time in our proposal, is a promising approach toward creating a new generation of trypanocides. However, a detailed molecular analysis of the polyadenylation process is required for validation of this enzyme as a drug target.